A primary goal of comparative genomics is to understand the forces that give rise to and maintain genetic variation in natural populations and among species. In an effort to understand the natural variation found in Drosophila mitochondrial genomes, I propose to examine sequence variation within natural populations and among different species to determine the factors influencing the levels of variation. This will be accomplished by testing the observed genomic sequence variation in several separate ways: 1) To determine whether the mitochondrial genome of Drosophila undergoes genetic recombination; 2) To determine whether mitochondrial genome sequence variation observed within Drosophila populations is due to the accumulation of neutral mutations, or the result of mutational substitutions that are maintained by natural selection; 3) To determine the factors affecting mitochondrial genome sequence variation in these genes among different species of Drosophila, such as functional constraints of amino acid substitution; and 4) To determine if the mitochondrial genome sequence variation among genes is due to asymmetrical directional mutation pressure. These questions will be tested by sequencing the mitochondrial genomes of six species of Drosophila (25 samples per species), and of another 52 species of Drosophilia (1 sample per species).